Kamrat Thomas, Nidetzky Bernd
Research Centre Applied Biocatalysis, Petersgasse 14, c/o Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria.
J Biotechnol. 2007 Mar 30;129(1):69-76. doi: 10.1016/j.jbiotec.2006.11.020. Epub 2006 Dec 2.
beta-Glycosidase CelB from the hyperthermophilic archaeon Pyrococcus furiosus is a versatile biocatalyst that has been used for the hydrolysis and synthesis of beta-d-glycosidic compounds at high temperatures and in non-conventional solvents. In spite of its outstanding thermal stability, CelB is prone to inactivation in the presence of reducing sugars and through recirculation in loop enzyme reactors. Entrapment into E. coli cells was used here to improve the stability of recombinant CelB under conditions promoting strong inactivation. Glutardialdehyde-mediated protein cross-linking or rigidification of the cell membrane by adding magnesium ions was required to prevent release of CelB from within the cell into the bulk solution. In the presence of 1M glucose or when applying recirculation rates of 2.6 min(-1), the entrapped enzyme was around two-fold more stable at 80 degrees C than free CelB. The significance of the stabilisation was attenuated by the decrease in CelB initial activity which was due to cross-linking and glutardialdehyde concentration-dependent. Entrapment facilitated downstream processing of CelB and biocatalyst recovery in repeated batchwise conversions of lactose at elevated temperatures.
来自嗜热古菌激烈火球菌的β-糖苷酶CelB是一种多功能生物催化剂，已被用于在高温和非传统溶剂中水解和合成β-D-糖苷化合物。尽管CelB具有出色的热稳定性，但在存在还原糖的情况下以及在循环酶反应器中循环时，它容易失活。在此使用包埋到大肠杆菌细胞中来提高重组CelB在促进强烈失活的条件下的稳定性。需要戊二醛介导的蛋白质交联或通过添加镁离子使细胞膜硬化，以防止CelB从细胞内释放到本体溶液中。在存在1M葡萄糖的情况下或当应用2.6 min⁻¹的循环速率时，包埋的酶在80℃下的稳定性比游离CelB高约两倍。由于交联和戊二醛浓度依赖性导致CelB初始活性降低，从而减弱了稳定化的意义。在高温下乳糖的重复分批转化中，包埋促进了CelB的下游加工和生物催化剂的回收。
